Dissolution guided wetting of structured surfaces

ABSTRACT

A microfabricated device having at least one gas-entrapping feature formed therein in a configuration that entraps air bubbles upon wetting the feature with a solvent or solution is described. The device includes a sacrificial residue in contact with the gas-entrapping feature, the dissolution of which guides the wetting of the gas-entrapping feature.

CROSS-REFERENCES

This application is a continuation application of U.S. patentapplication Ser. No. 15/995,360, filed Jun. 1, 2018, which is acontinuation application of U.S. patent application Ser. No. 14/404,225,filed Nov. 26, 2014, now U.S. Pat. No. 9,994,805, which was filed underthe provisions of 35 US.C. § 371 and claims the priority ofInternational Patent Application No. PCT/US2013/043562, filed May 31,2013, which is related to U.S. provisional application No. 61/653,783,filed May 31, 2012, the contents of all of which are incorporated hereinby reference in their entirety.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under Grant NumbersEB012549 and HG004843 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

FIELD OF THE INVENTION

The present invention concerns microfabricated devices and methods ofwetting gas-entrapping features therein.

BACKGROUND OF THE INVENTION

Lab-on-a-chip technology has made rapid progress for applications incell biology and biochemical assay.¹ Lab-on-a-chip systems that enablethe efficient performance of assays with low reagent consumptiontypically contain features on the structured surfaces of themicrofabricated devices such as microfluidic chips and microwell arrayswhere air bubbles can be easily trapped upon the addition of a solventor solution. The trapped air bubbles result in Cassie-state wetting onthe surface. This wetting phenomenon has been exploited for specificapplications, such as selective deposition of proteins and cells to theareas that are in contact with the aqueous solution, for example, on thesurface between microwells (but not inside microwells),⁷ or on the topsurface of micropallets (but not in the space among micropallets).⁸Nevertheless, for a majority of applications, the trapped air bubbles inmicrofabricated devices are an obstacle in the use of the device and theair bubbles need to be removed to allow the entire surface to be in fullcontact with solutions of analytes, cell-culture medium, or otherfluids.^(9, 10)

Microwell arrays, useful platforms for cell culture and assays atsingle-cell resolution, are examples of microfabricated devicespossessing gas-entrapping features.^(2, 11) Since microwell arrays areoften made from polymers, such as PDMS, which are either hydrophobic oronly slightly hydrophilic in their native form, trapping of air bubblesinside the microwells are encountered whenever the array is covered withan aqueous solution. To solve this problem, plasma treatment isgenerally used to make the surface hydrophilic; however, in many of thecommon polymers this hydrophilization is only temporary, and either apartial or complete hydrophobic recovery is usually observed.^(14, 15)In addition to surface oxidation, several methods are currently used forremoving trapped air bubbles in cavities, including vacuum application,pressurization, centrifugation, vibration and sonication.^(10, 16-18)Alternatively, low surface tension liquids (e.g. ethanol, γ=22.4 mN·m⁻¹can be used to initially wet the surface prior to exchange with water(γ=72.9 mN·m⁻¹) or an aqueous buffer.^(19, 20) Besides microwell arrays,corners and dead ends in microfluidic channels have similar problemswith surface wetting and bubble formation. A new microfluidic design,called a phaseguide, based on a step-wise advancement of the liquid-airinterface using the meniscus pinning effect, can effectively eliminatethe probability of trapping air bubbles in complex microfluidicgeometries such as corners and deep angular structures.⁶ However, thismethod is difficult to remove trapped air in microcavities microwells,or dead ends, since it relies on the creation of strips of material onthe wall along the direction of advancing fluid.

Although all of the above methods are effective in preventing orremoving air bubbles in specific cases, there remains a need for asimpler, passive method for preventing the formation of gas bubbles orremoving gas bubbles from microfabricated devices having microcavities,corners, dead ends and other gas-entrapping features.

SUMMARY OF THE INVENTION

A first aspect of the invention is a microfabricated device (e.g., amicrowell array, a microfluidic device) having at least onegas-entrapping feature on a structured surface formed therein thatentraps gas bubbles which prevent the wetting of said feature with asolvent or solution. The device includes a sacrificial residue incontact with said gas entrapping feature. The nature of the sacrificialresidue may be either hydrophilic or hydrophobic, and may be either asolid or a combination of a solute and solvent suitable for thegas-entrapping feature to be wetted.

In some embodiments, the gas-entrapping feature comprises a microwell,corner, microcavity, dead end, post, trap, hole, passage, channel, orcombination thereof.

In some embodiments, the surface of the gas entrapping feature isoxidized (e.g., plasma oxidized).

A further aspect of the invention is a method of wetting amicrofabricated device while inhibiting the entrapment of gas bubblestherein, comprising: (a) providing a microfabricated device as describedherein, (b) treating the microfabricated device by priming with asacrificial residue in contact with the gas-entrapping features, andthen (c) treating said microfabricated device with a solvent or solutionsufficient to dissolve and remove said sacrificial residue from saidgas-entrapping feature while concurrently wetting said gas-entrappingfeature with said solvent or solution.

The foregoing and other objects and aspects of the present invention areexplained in greater detail in the drawings herein and the specificationset forth below. The disclosures of all United States patent referencescited herein are incorporated by reference herein in their entirety.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 . Wetting of PDMS microwells. (A) Brightfield images of PDMSmicrowells (H=55 μm, D=50 and 100 μm) placed in an aqueous solution atday 0, day 3 and day 6 after plasma treatment. Air bubble entrapment waspresent on day 3 (50 μm) and day 6 (100 μm). (B) Schematic showing thewetting of microwells. ϕ is defined as the angle that a diagonal throughthe well makes with the well's base and top edge of its side wall and θis the angle of the aqueous solution on the side wall of the cavity.

FIG. 2 . Priming the hydrophilic microwells with glucose. SEM andbrightfield images showing a microwell (D=200 μm, H=55 μm) filled with aglucose solution and then dried. The volumetric concentration of glucosewas varied as shown in the figure. SEM images were obtained at a tiltangle of 30°.

FIG. 3 . Dissolution guided wetting in microwells. (A) Schematic showingthe wetting process on a microwell guided by dissolution of glucose. (B)Wetting in PDMS microwells (D=50 μm, H=55 μm) with 37% glucose priming(left panel) and without glucose priming (right panel). Prior towetting, PDMS samples were treated with air plasma for 2 min., primedwith 37% glucose (or not primed), and stored at room temperature in airfor one month. (C) Time-lapse fluorescence images showing thedissolution of glucose (mixed with 200 μg/mL TRITC dextran) in amicrowell array (D=200 μm, H=55 μm).

FIG. 4 . Dissolution guided wetting in corners and dead ends ofmicrofluidic channels. (A) Air bubble entrapment was present in corners(i) and dead ends (ii) of PDMS microfluidic channels on day 7 afterbonding. (B) Priming the corners (i, ii) with glucose, and dead ends(iii, iv) with sorbitol. Transmitted light (i and iii) and fluorescence(ii and vi) images clearly show the corners and dead ends were occupiedwith sugar. The sugar was mixed with 200 μg/mL TRITC dextran forfluorescence imaging. (C) Time-lapse transmitted light images showingthe dissolution of glucose in corners (i) and sorbitol in dead ends(ii). Arrows indicate the direction of water flow. Prior to test, thechannels were stored at room temperature in air for one week. Scalebar=50 μm.

FIG. 5 . Schematic showing that the drying of glucose solution in amicrowell results in a conformal, elliptical, cone-shaped coating ofsolid glucose. The degree of coverage depends on the concentration ofglucose.

FIGS. 6A and 6B. ATR-FTI R spectra of PDMS samples primed with glucose(FIG. 6A) and sorbitol (FIG. 6B) then rinsed with water. A native PDMSsample was used as a negative control, and glucose and sorbitol wereused as positive controls.

FIG. 7 . Transmitted light images showing glucose guided wetting inmicrowells formed in polystyrene. Scale bar=100 μm

FIG. 8 . Transmitted light images showing sorbitol guided wetting inPDMS microwells. Scale bar=100 μm.

FIG. 9 . Process of priming the corners and dead ends of microfluidicchannels with a 30 vol % glucose solution or a 50 vol % sorbitolsolution.

FIG. 10 . Shape of glucose residue in PDMS microwells is dependent onthe interfacial property of PDMS/glucose solution. (A) Schematic showingthe wetting of glucose solution in a PDMS microwell upon drying. Thecontact angle θ determines the shape of glucose. (B) A parabolic residueof glucose is formed on an oxidized, hydrophilic PDMS. (C) A flat,column shaped residue of glucose is formed on a native, hydrophobicPDMS.

FIG. 11 . Wetting of microraft array without (A) and with (B) depositionof glucose residue. (i) Schemes of cross-sectional view of a raft. (ii)Schemes of wetting on a raft. (iii) Transmitted light images showingwetting on a raft array. Each well has a dimension of 200 μm×200 μm×100μm (length×width×height). To deposit glucose residue on the array, a 40wt % glucose solution was applied on the raft array followed byaspiration to remove excess solution and drying in air.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is now described more fully hereinafter withreference to the accompanying drawings, in which embodiments of theinvention are shown. This invention may, however, be embodied in manydifferent forms and should not be construed as limited to theembodiments set forth herein; rather these embodiments are provided sothat this disclosure will be thorough and complete and will fully conveythe scope of the invention to those skilled in the art.

Like numbers refer to like elements throughout. In the figures, thethickness of certain lines, layers, components, elements or features maybe exaggerated for clarity. Where used, broken lines illustrate optionalfeatures or operations unless specified otherwise.

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of the invention. Asused herein, the singular forms “a,” “an” and “the” are intended toinclude plural forms as well, unless the context clearly indicatesotherwise. It will be further understood that the terms “comprises” or“comprising,” when used in this specification, specify the presence ofstated features, integers, steps, operations, elements components and/orgroups or combinations thereof, but do not preclude the presence oraddition of one or more other features, integers, steps, operations,elements, components and/or groups or combinations thereof.

As used herein, the term “and/or” includes any and all possiblecombinations or one or more of the associated listed items, as well asthe lack of combinations when interpreted in the alternative (“or”).

Unless otherwise defined, all terms (including technical and scientificterms) used herein have the same meaning as commonly understood by oneof ordinary skill in the art to which this invention belongs. It will befurther understood that terms, such as those defined in commonly useddictionaries, should be interpreted as having a meaning that isconsistent with their meaning in the context of the specification andclaims and should not be interpreted in an idealized or overly formalsense unless expressly so defined herein. Well-known functions orconstructions may not be described in detail for brevity and/or clarity.

It will be understood that when an element is referred to as being “on,”“attached” to, “connected” to, “coupled” with, “contacting” or “incontact” with, etc., another element, it can be directly on, attachedto, connected to, coupled with and/or contacting the other element orintervening elements can also be present. In contrast, when an elementis referred to as being, for example, “directly on,” “directly attached”to, “directly connected” to, “directly coupled” with or “directlycontacting” another element, there are no intervening elements present.It will also be appreciated by those of skill in the art that referencesto a structure or feature that is disposed “adjacent” another featurecan have portions that overlap or underlie the adjacent feature.

Spatially relative terms, such as “under,” “below,” “lower,” “over,”“upper” and the like, may be used herein for ease of description todescribe an element's or feature's relationship to another element(s) orfeature(s) as illustrated in the figures. It will be understood that thespatially relative terms are intended to encompass differentorientations of the device in use or operation in addition to theorientation depicted in the figures. For example, if the device in thefigures is inverted, elements described as “under” or “beneath” otherelements or features would then be oriented “over” the other elements orfeatures. Thus the exemplary term “under” can encompass both anorientation of over and under. The device may otherwise be oriented(rotated 90 degrees or at other orientations) and the spatially relativedescriptors used herein interpreted accordingly. Similarly, the terms“upwardly,” “downwardly,” “vertical,” “horizontal” and the like are usedherein for the purpose of explanation only, unless specificallyindicated otherwise.

It will be understood that, although the terms first, second, etc., maybe used herein to describe various elements, components, regions, layersand/or sections, these elements, components, regions, layers and/orsections should not be limited by these terms. Rather, these terms areonly used to distinguish one element, component, region, layer and/orsection, from another element, component, region, layer and/or section.Thus, a first element, component, region, layer or section discussedherein could be termed a second element, component, region, layer orsection without departing from the teachings of the present invention.The sequence of operations (or steps) is not limited to the orderpresented in the claims or figures unless specifically indicatedotherwise.

Devices of the present invention are, in general, microfabricateddevices such as microwell arrays and microfluidic devices formed from orin a selected substrate. Such devices are known and examples include,but are not limited to, those described in U.S. Pat. Nos. 7,927,830;7,775,088; 7,742,662; 7,556,776; 7,169,577; 7,161,356; 6,670,133; and6,632,655. In some embodiments, the microfabricated devices have one ormore fluid passages, chambers, channels, wells, conduits or the likethat are configured to contain microvolumes of liquids, typicallywherein one or more of the dimensions is less than 500 μm. In someembodiments, the device further comprises a main channel in fluidcommunication with the gas-entrapping feature; wherein the surface ofsaid main channel is substantially free of the sacrificial residue. Insome embodiments, the device comprises a microfluidic network, with thegas-entrapping feature comprising a first region of the microfluidicnetwork.

Devices of the invention can be formed from any suitable substratematerial including but are not limited to silica based substrates, suchas glass, quartz, silicon or polysilicon, as well as other substratematerials, such as gallium arsenide and the like. Other suitablematerials include but are not limited to polymeric materials, e.g.,plastics, such as polymethylmethacrylate (PMMA), polycarbonate,polytetrafluoroethylene (e.g., TEFLON™) polyvinylchloride (PVC),polydimethylsiloxane (PDMS), polysulfone, polystyrene, polycarbonate,polyimide, cyclic-olefin copolymer, polymethylpentene, polypropylene,polyethylene, polyvinylidine fluoride, acrylonitrile-butadiene-styrenecopolymer (ABS), and the like, as well as polymerized photoresists,e.g., SU-8, 1002F and the like (see, e.g., U.S. Pat. No. 6,103,446).

Substrate materials are often selected based upon their compatibilitywith known techniques, such as microfabrication. Suitable substratematerials are also generally selected for their compatibility with thefull range of conditions to which the microfabricated. devices may beexposed, including extremes of pH, temperature, salt concentration, andapplication of electric fields. Accordingly, the substrate material mayinclude materials normally associated with the semiconductor industry.In the case of semiconductive materials, it will often be desirable toprovide an insulating coating or layer, e.g., silicon oxide, over thesubstrate material, and particularly in those applications whereelectric fields are to be applied to the device or its contents. In someembodiments, the substrates used to make the mircofabricated device aresilica-based, more preferably glass or quartz, due to their inertness tothe conditions described above, as well as the ease with which they aremicrofabricated in other embodiments polymeric substrate materials arepreferred for their ease of manufacture, low cost and disposability, aswell as their general inertness to most extreme reaction conditions.These polymeric materials may include treated surfaces, e.g.,derivatized or coated surfaces, to enhance their utility in themicrofluidic system, e.g., provided enhanced fluid direction, e.g., asdescribed in U.S. Pat. No. 5,885,470, and which is incorporated hereinby reference in its entirety for all purposes. In other embodiments themicrofabricated device is made using a combination of materials, such assilica-based and polymeric materials.

The material of the microfabricated device may be opaque, translucent ortransparent. The device can be formed of a single layer substrate of asingle material or a laminated or multi-layer configuration of the sameor different material substrates. The device may be a single layermonolithic substrate or (more typically) a multiple layer device (e.g.,having two or three layers or more) and having a thickness that isbetween about 0.2 mm to about 15 mm. The thickness of the device is notcritical, as the thickness of top and bottom parts of the device are notcritical, so long as the proper inner chamber dimensions are providedfor the intended use. The device can comprise a bioactive agent that isformed in a matrix of the substrate and/or applied or coated on aprimary surface thereof to define one or more analytical sites on thedevice for analysis and/or to define a barrier zone.

Microfabricated devices of the invention can be made by any suitabletechnique including but not limited to microfabrication techniques suchas photolithography, wet chemical etching, laser ablation, reactive ionetching (RIF), air abrasion techniques, injection molding, LIGA methods,metal electroforming, embossing, and other techniques. Suitabletechniques may also be those employed in the semiconductor industry.Other suitable techniques include but are not limited to moldingtechniques, such as injection molding, embossing or stamping, or bypolymerizing the polymeric precursor material within the mold (See U.S.Pat. No. 5,512,131).

Microfabricated devices may have either hydrophobic or hydrophilicsurfaces. Further, hydrophobic surfaces can be made hydrophilic bytreatment with air or oxygen plasma, chemical surface modification, orphysical surface deposition. For example, PDMS is a hydrophobic materialand its hydrophobic surface can be made hydrophilic by oxygen plasmatreatment (See Bodas D. and Khan-Malek, C. Microelectronic Engineering,2006, 83, 1277-1279), or by chemical grafting of a thin hydrophilicpoly(acrylic acid) on a hydrophobic PDMS surface (See U.S. Pat. No.6,596,346 and Analytical Chemistry, 2002, 74, 4117-41.23); or byphysical deposition of a glass layer (See Abate, A. et al., Lab Chip,2008, 8, 516-518).

Microfabricated devices frequently contain gas-entrapping features suchas microcavities, dead ends, corners, posts, holes, channels, traps, andpassages. As used herein, gas refers to any substance in the gaseousphase and may include nitrogen, oxygen, carbon dioxide, or mixtures suchas air.

The microfabricated devices are primed with a sacrificial residue thatis suitable for dissolution guided wetting of the gas-entrappingfeatures and therefore remove or-prevent the formation of gas bubbles inthe device. For microfabricated devices that use solvents or solutionsthat are aqueous, the sacrificial residue may be comprised of anysuitable material, including but not limited to salts, carbohydrates(e.g., monosaccharide, disaccharide, oligosaccharide, orpolysaccharide), and other hydrophilic polymers. Particular examplesinclude, but are not limited to, sodium chloride, dextran, polyethyleneglycol, poly(acrylic acid), poly(4-vinylpyridine), poly(vinyl alcohol),dextran, alginate, agarose, chitosan, cellulose, glucose, sucrose andsorbitol. In some embodiments, the sacrificial residue is comprised of anon-metabolizable sugar (e.g., sorbitol, xylitol or mannitol, etc.).When using a salt as the sacrificial residue, a salt that is readilydissolved in an aqueous solution may be used. Non-limiting examples ofsuch a salt are sodium chloride, potassium chloride, sodium sulfate,sodium bisulfate, sodium phosphate, monosodium phosphate, disodiumphosphate, potassium phosphate, monopotassium phosphate, dipotassiumphosphate, calcium chloride, magnesium chloride, or a combinationthereof. The sacrificial residue is, in some embodiments, amorphous.When a microfabricated device uses a solvent or solution that isnon-aqueous, then a suitable material that is hydrophobic should bechosen for the sacrificial residue. Such suitable hydrophobic materialsare hydrophobic polymers, low molecular weight organic solids,non-volatile liquids. Particular examples of hydrophobic polymersinclude, but are not limited to, polyethylene, polypropylene, polyvinylchloride, polystyrene, nylons, polyesters, acrylics, polyurethane, andpolycarbonates, polylactic acid, poly(lactic-co-glycolic acid),poly(methyl methacrylate). Particular examples of low molecular weightorganic solids include, but are not limited to, paraffin wax,naphthalene, anthracene, aspirin (acetylsalicylic acid), 2-naphthol,fat. Particular examples of non-volatile liquids include, but are notlimited to, synthetic oils (for example mineral oil), vegetable oils(for example olive oil), lipids, silicone oil, liquid epoxy resin.

The sacrificial residue may be applied by any suitable technique. In oneembodiment, the sacrificial residue is a suitable solute that isdissolved in an aqueous or non-aqueous solvent, contacting the selectedsolution comprising the solvent and solute with the gas-entrappingfeature to be primed, and allowing the solution to dry thereon therebydepositing the solute in contact with the gas-entrapping feature. Inanother embodiment, a material for the sacrificial residue may bedispersed, rather than dissolved, in a suitable aqueous or non-aqueoussolvent. In yet another embodiment, the sacrificial layer is comprisedof a solid material that may be brought into contact with agas-entrapping feature as a fine particulate dust and/or by melting thesolid at a suitable temperature.

It is noted that the sacrificial residue used to achieve dissolutionguided wetting of the structured surface does not chemically modify thesurface. While plasma oxidation of the PDMS surface can be used incertain embodiments, this step to modify the surface is not required asthe application of vacuum or solvents with the appropriatecharacteristics could be used to provide Wenzel-state wetting of thestructured surface in the deposition of the sacrificial residue incontact with gas-entrapping features.

Once prepared, the primed microfabricated devices may be packaged in awater-proof container, and/or packaged in a container with a desiccant,for subsequent use.

In use, the primed microfabricated devices are typically rinsed,depending on its intended purpose, with an aqueous or non-aqueous rinsesolution for a time, in an amount and at a temperature sufficient todissolve and remove the sacrificial residue from said gas entrappingfeature, and preferably while concurrently wetting said gas entrappingfeature with said rinse solution. The device may then be rinsed with asecond aqueous or non-aqueous solution. (e.g., a growth media, an assayor reagent media, a reaction media, etc.) to remove the previous rinsesolution therefrom and ready the device for its intended purpose.

The present invention is explained in greater detail in the followingnon-limiting examples.

A method is described to prevent or eliminate gas bubbles on structuredsurfaces of microfabricated devices possessing microcavities, corners,dead ends and other gas-entrapping features. In one example, amicrofabricated device was made using PDMS. The process for priming themicrofabricated device for dissolution guided wetting of gas-entrappingfeatures was composed of two steps: the surface was first madehydrophilic by plasma treatment, then primed with an aqueousmonosaccharide solution, and placed in dry storage. Prior to use of themicrofabricated device, the end user simply adds water to dissolve amonosaccharide followed by rinsing to remove any trace of themonosaccharide in the microfabricated device or in solution. Thedissolution of the monosaccharide guides a complete wetting of thegas-entrapping features, leaving the structured surfaces free of gasbubbles. Microwells and microfluidic channels made from PDMS were usedas the model to demonstrate this dissolution-guided wetting of thegas-entrapping features.

Materials used were D-glucose, D-sorbitol, phosphate buffered saline(PBS) tablets, tetramethylrhodamine isothiocyanate-dextran(TRITC-dextran, average molecular weight 500,000), andoctyltrichlorosilane were purchased from Sigma Aldrich (St. Louis, Mo.).SU-8 photoresist was purchased from MicroChem Corp. (Newton, Mass.).PDMS was prepared from the Sylgard 184 silicone elastomer kit (DowCorning, Midland, Mich.).

PDMS, a polymer known to have a rapid and complete hydrophobicrecovery,¹⁵ was selected as the material to create the structuredsurfaces of the microwells and microfluidic channels. Microwell arraysand microfluidic channels were fabricated by micromolding PDMS on anSU-8 master by conventional soft lithography.²¹ The SU-8 master wasfabricated by standard photolithography on a glass slide spin-coatedwith an SU-8 layer of 55 μm thickness.²² The master mold was treatedwith 50 μL, oetyltrichlorosilane in a vapor-phase silanization processin a polycarbonate desiccator (Fisher Scientific): the desiccator wasdegassed by an oil-free pump for 2 min and then closed for 16 h. PDMSprepolymer (10:1 mixture of base: curing-agent in the Sylgard 184 kit)was spread on the master mold and degassed under vacuum to remove airbubbles from the polymer. The master was baked at 100° C. on a hotplatefor 30 min to cure the PDMS. The PDMS forming the microwell arrays ormicrochannels was then obtained by peeling it from the master. Formicrowell arrays, the depth of the microwells was 55 μm, and thediameter was in the range from 10 μm to 3 mm. For microfluidic channels,holes of 2-mm diameter were first punched at the ends of the patterns onPDMS to serve as solution reservoirs. The PDMS and a glass slide weretreated in an air-plasma cleaner (Harrick Plasma, Ithaca, N.Y.) for 2min before they were sealed to form an enclosed microfluidic channel.

The degree of wetting of the gas-entrapping features was determined bymeasuring the water contact angle. The water contact angle was measuredwith a pocket Goniometer PG-3 (Fibro system AB, Sweden) using a 5 μLdrop of deionized water. The contact angle was measured at 5 s after thedrop was applied. An average of 10 measurements was calculated persurface.

Microwells were primed with glucose as the sacrificial residue. Glucosesolutions in water of different volumetric concentrations (0%, 22%, 30%,and 37%) were prepared. The volumetric concentration of glucose wascalculated from its weight concentration by assuming the volume ofsolution is the sum of the volumes of the solute and solvent. PDMSmicrowell arrays were first treated in an air-plasma for 2 min togenerate hydrophilic surfaces by oxidation. An open chamber was createdsurrounding the array using a self-sealing, square PDMS ring (25×2.5×6mm) attached to the substrate. An aqueous solution of glucose (1-2 mL)was added to the chamber to wet the array surface. The array was tilted,and the excess solution was removed by aspirating with a-pipet attachedto a vacuum hose and liquid trap. By evaporating the remaining water atroom temperature in air, solid glucose was deposited in the. microwells.The microwells primed with glucose were observed with. a Nikon EclipseTE300 inverted, fluorescence microscope and by scanning electronmicroscopy (SEM) (FEI Quanta 200 ESEM, FEI Company).

FIG. 9 shows the protocol for priming the corners and dead ends ofmicrofluidic channels. Immediately after plasma treatment and bonding onglass, 5 μL of the solution was added to one entrance of themicrofluidic channel. Due to the hydrophilic surface of the freshlyplasma-bonded PDMS, the solution spontaneously wet the entire channelwithin a few minutes, even in the corners and dead ends without anytrapped air bubble. A hose supplied with nitrogen was placed on theother entrance of the microfluidic channel to purge the main channel, sothat the solution in the main channel was expelled leaving residualsugar solution in the corners and dead ends. The solution flushed intothe reservoir was removed by aspiration. A drop of water was added tothe entrance while the main channel was being. purged with nitrogen. Thewater diluted the expelled sugar solution at the entrance and wasquickly aspirated, which serves to prevent the possibility of driedsugar solution blocking the main channel. After priming, the deviceswere stored at room temperature. The sugar solution trapped in cornersand dead ends within the device was allowed to gradually dry over 1-2days by evaporation. A 30 vol % glucose solution was used to fill thecorners in a microfluidic channel. A 50 vol % sorbitol solution was usedto fill the dead ends in a microfluidic channel where sorbitol was foundto be more effective due to the higher solubility of sorbitol in water.

The wetting behavior (complete wetting, partial wetting, or non-wetting)of PDMS microwell arrays and microfluidic channels, either primed withglucose or not, was determined by adding water and observing thepresence of trapped air bubbles under microscope. Air bubbles in themicrowells and microfluidic channels were readily discerned bybrightfield microscopy by virtue of a thick dark boundary being formedbetween air and PDMS due to differences in the refractive indices ofwater (1.33), PDMS (1.43)²³ and air (1.00),⁷ Similarly, deposition ofglucose in the microwells and microfluidic channels could be ascertaineddue to refractive index mismatches of air (1.00), PDMS (1.43) andglucose (1.51).²⁴ To observe the dissolution process of glucose byfluorescence microsocopy, the glucose solution was mixed with 200. μg/mLTRITC-dextran and used to prime microwells and microfluidic channels.The microwells and microfluidic channels were imaged using the NikonEclipse TE300 microscope equipped with a CY3 filter set (0-2E; NikonInstruments; excitation filter 528-553 nm dichroic 565 nm long pass,emission 590-650 nm). Time lapse images were collected with a cooled CCDcamera (Photometrix Cool Snap; Roper Scientific, Tucson, Ariz.) usingNIS-Elements software.

Air bubbles are often trapped in microfabricated devices. For a majorityof biological applications, full wetting of microwells, i.e. Wenzelwetting state, without entrapment of air bubbles is often a requirement.Since many polymers (such as PDMS) used for these microfabricateddevices are hydrophobic, entrapment of air within the hydrophobicmicrowell cavity is a frequent: occurrence unless additional steps aretaken⁷ To prevent air bubble formation, plasma treatment, either oxygenor air, is often used to render the microwell surface hydrophilic andpermit instantaneous aqueous wetting (FIG. 1A).²⁵ However, for manypolymers, the surface recovers its hydrophobic state quickly afteroxidation so that the devices cannot be re-wetted after long-termstorage without re-oxidation of the device.²⁶ Among ten differentpolymers of interest air microfabrication, PDMS has the most rapidhydrophobic recovery.¹⁵ In the current studies, the water-dropletcontact angle on PDMS films immediately after plasma treatment was10°±5°, but recovered to 42°±8° (n=3) after 3 days. The microwell arraycould be fully wetted on day 0, but by day 3 air bubbles were trappedinside small microwells (diameter D=50 μm, height H=55 μm, FIG. 1A) uponaddition of water. The water-droplet contact angle on the PDMS surfacecontinued to increase over time to 65°±11° (n=3) at day 6 withentrapment of air bubbles even in large microwells (D=100 μm, H=55 μm,FIG. 1A). A similar trend was observed for microwells made from otherpolymers, such polystyrene, poly(D,L-lactide) and SU-8 epoxyphotoresist, although these exhibited a slower hydrophobic recovery thanPDMS. These observations are consistent with a recent report whichtested hydrophobic recovery of 10 common polymers.¹⁵

The stability or duration of air trapping inside the wells was alsodependent on the hydrophobicity of the PDMS. When the contact anglerecovered to 42°±8° (n=3) at day 3, the air bubbles persisted forapproximately 10 min inside the microwells (D=50 μm, H=55 μm) beforedisplacement by water. When the contact angle recovered to 75°±5° (n=3)at day 14, the air bubbles were stably trapped in the microwells for >4hours (D=50 μm, H=55 μm). For hydrophilic materials (contact angle <90°)with cavities, trapping of air is thought to be possible—since thecavities can provide an energy barrier to stop the water fromentering—them, Wenzel-state wetting is thermodynamically favorable forhydrophilic materials (contact angle <90°) with cavities,²⁷ but thisenergy barrier may be adequate to prevent, at least temporarily, thetransition from a Cassie state to a Wenzel state. For example, aspreviously reported, air can be trapped in spherical cavities 400-800 nmin diameter on hydrophilic gold surfaces (contact angle=700°.²⁸

The trapping of air bubbles inside microwells can be explained by thescheme shown in FIG. 1B where is defined as the angle that a diagonalthrough the well makes with the well side wall and θ is the angle of theaqueous solution on the side wall of the cavity. For a hydrophilicsurface, such, as freshly oxidized PDMS, θ<ϕ, the advancing liquid canwet the vertical wall and bottom before reaching the other edge of thewell. As a result, air can be pushed out from the well resulting in a.homogeneous wetting (Wenzel state). For a hydrophobic surface, θ<ϕ,advancing liquid reaches the other edge of the well before it can wetthe side and bottom walls (FIG. 1B). As a result, air can be trappedinside the well resulting in heterogeneous wetting (Cassie state).Wetting of a rough solid surface with a liquid has been extensivelystudied with theoretical models, although the exact mechanism of airentrapment has not been elucidated.^(29, 30, 31, 32)

The priming of hydrophilic microwells with glucose can prevent thetrapping of air bubbles in microcavities through dissolution guidedwetting eliminates a significant annoyance in the use of microwellarrays for a standard biology lab. Typically, end users prefer to havedevices in a ready-to-use state without the need for pre-processingimmediately prior to the biological application. In the case ofmicrowell arrays, a fully wettable surface without the need for plasmaoxidation, high vacuum exposure, or pre-treatment with a toxic,low-viscosity liquid such as ethanol would facilitate their use andacceptance. The priming of gas-entrapping features with glucose, as oneexample of a sacrificial residue, achieves the goal of providing amicrofabricated device that could be stored for long periods, yet remainfully wettable. PDMS microwells were first oxidized with air plasma togenerate a hydrophilic surface. A solution of water-soluble material wasadded to the microwells forming Wenzel-state wetting on the surface.Excess liquid was removed by aspiration. Upon drying, a conformalcoating of solid material was generated inside the microwells. Water (ora suitable aqueous solution) could then fully wet the primed microwellsat a later time by dissolving the sacrificial residue of glucose insidethe microwells, thus preventing or eliminating air bubbles in themicrocavities. Although many water soluble materials are available, apreferred embodiment uses non-toxic, biocompatible materials with fastdissolution rates in water. Phosphate buffered saline (PBS), a commonsalt solution used in biology, may also be used but care should be takento avoid the formation of large salt crystals formed in the wells thatcan pop out of place instead of a conformal coating on the well walls.Solid sugar polyols, such as D-glucose and D-sorbitol, are preferredmaterials for the sacrificial residue as they rapidly dissolve in waterand are biocompatible. In one embodiment, a PDMS microwell array waswetted with an aqueous glucose solution (37% volumetric concentration)and the excess volume above the wells was then removed. After solventevaporation, a conformal coating of solid glucose lined the microwellwalls and floor (FIG. 2 ). For the microwells primed with glucose at aconcentration of 30%, the glucose layer appeared to coat only a portionof the microwell walls and floor (FIG. 2 ). Lower glucose concentrations(22%) resulted in even lower coverage of the microwell surface. Theangle of the glucose layer on the side wall was also steeper at thelower glucose concentrations. This observation is consistent with thetheoretical calculation by assuming that the contour of the driedglucose layer is regarded as elliptical in shape. The relative coverageof dry glucose in the microwell is dependent only on the concentrationof glucose c, not on the dimension of microcavities (diameter andheight). Therefore, when using glucose as the sacrificial residue toprime the microcavities a preferred embodiment is to use a glucosesolution with c>33%, for a structured surface possessing microcavitieswith a wide range of sizes (10 μm-3 μm) and depths.

The glucose coating alters both θ for the side wall and for the well,which facilities the rewetting of the microwells by water. Since watercan rapidly dissolve glucose, its dissolution guides the rewetting ofthe microwells (FIG. 3A), To determine if the change in θ and ϕ for theglucose-coated wells could result in microwell wetting, PDMS microwellarrays (D=50 μm, H=55 μn) were tested one month after oxidation andpriming with a 37% or 0% glucose solution. FIG. 3B shows the wetting onPDMS microwells with glucose priming (left panel) and without glucosepriming (right panel). For microwells without glucose priming, airbubbles formed in microwells. For microwells primed with a 37% glucosesolution, no air bubbles formed. This anti-bubble function was effectivefor 4 months, the longest storage time tested to date, suggesting thatthe changes in θ and ϕ were successful in maintaining wettability overtime. To visualize the rate of glucose dissolution, a fluorescentlydoped glucose solution (37% glucose with 200 μg/mL TRITC-dextran, 0.5MD) was used to prime a PDMS microwell array (microwells with D=200 μm,H=55 μm) one month prior to the experiment. The loss of fluorescence inthe well was then tracked over time to follow glucose dissolution. Afteraddition of water, the microwells were observed to completely wetfollowed by a loss of the priming layer over a time period of 25 s (FIG.3A, C). Following wetting of the well's top surface, dissolution ofglucose guides the filling of the well with the aqueous solution. Videoexamination showed the difference in the wetting characteristics ofmicrowells with and without glucose coating.

A source of the change in θ is the presence of the glucose coating whichcreates a hydrophilic sacrificial residue in contact with thegas-entrapping features of the PDMS microfabricated device such that thewater rapidly and entirely wets the structured surface of the device.However, it is possible that the glucose layer also prevents thehydrophobic recovery of the PDMS, thus facilitating the spread of theaqueous solution into the cavity near the edges of the glucose layer. Todetermine whether a glucose layer could maintain the hydrophilicity ofthe plasma-treated PDMS surface, the contact angle of PDMS films wasevaluated at varying times after priming with glucose. PDMS films wereoxidized with plasma and primed with 37% or 0% glucose solutions spreadover the surface and dried in air. The films were then stored in air for0, 2, 5, or 12 days (n=3 for each condition and time). Immediately priorto measurement of the water-droplet contact angle, the arrays wererinsed with water and dried under a nitrogen stream, and the contactangle was measured. An attenuated total reflectance (ATR)-FTIRspectrometer (Nicolet iSIO, Thermo Scientific) was used to confirm theabsence of glucose residue on the PDMS surfaces (FIG. 6 ). At day 0, thecontact angle for both primed and unprimed PDMS films was 10°±5°. By day2, the contact angle for glucose-primed and unprimed PDMS films was49°±2° and 41°−±6°, respectively. At day 5, the angle was 56°±4° for aprimed film and 63⁻°±6° for an unprimed film. At day 12, the angle was65°±8° for a primed film and 72°±4° for an unprimed film. These resultsdemonstrated that the glucose priming did not significantly delayhydrophobic recovery of the underlying PDMS structured surface. Theinitial change in θ<ϕ created by the glucose layer enabled wetting ofthe upper cavity surface while the subsequent dissolution of glucoseensured that the entire PDMS microcavity could be filled with theaqueous solution. This method for preventing bubble entrapmentfunctioned equally well for microwells made from other materials such aspolystyrene (FIG. 7 ). Sorbitol was also tested as a sacrificial residuefor priming PDMS microwells and results similar to those shown abovewere obtained (FIG. 8 ).

Similar to microwell arrays, air bubbles can be trapped in corners anddead ends of microfluidic channels (FIG. 4A). To demonstrate thedissolution guided wetting in the corners and dead ends of microfluidicchannels, microfluidic devices were built by molding PDMS channels froma master and then bonding it with glass slides through plasma oxidation.Immediately after bonding the channel was primed with glucose orsorbitol solution. Due to the hydrophilic nature of the freshly oxidizedPDMS surface, both the sugar solutions wet the entire channel quickly,even in corners and dead ends. The sugar solution was removed by purgingthe channel with nitrogen and aspiration from the reservoir. Afterpurging, residual sugar solution remained trapped in the corners anddead ends. A 30% glucose solution was used to prime the corners, and a50% sorbitol solution was used to prime dead ends. The higher solubilityof sorbitol (59.6 vol %) over glucose (37.1 vol %) was found to bepreferred for the dead ends used in these studies due to the smallvolumes trapped in these structures. Upon drying, residual solid sugarremained in the corners and dead ends (FIG. 4B). The priming of solidsugars is expected to survive for prolonged times due to theirnon-volatility, and their stability in a wide range of temperature dueto their high melting point (146° C. for glucose and 95° C. forsorbitol). After storing the devices at room temperature for 7 days,water was introduced into the channels (FIG. 4C). For the devicestudying air trapping in corners, water passing through the channeldissolved the glucose in the corners over a 30 s time period. Thedissolution of glucose was seen to guide the wetting of the corners in amanner similar to that seen in the microwells. A similar observation wasmade for the sorbitol-primed dead ends. Due to relatively smallerdimension (50×50 μm for the dead end) compared to corners (500 μmsquare), the dissolution was faster (˜10 s). Air bubble entrapment wasnot observed in the primed devices, but was present in identicalunprimed devices.

The coverage of glucose priming in a microwell may be estimated by theconcentration of the glucose solution used. Assuming the height of theliquid layer above the rim of the microwell was negligible, the totalvolume of glucose solution loaded into each microwell was:

$\begin{matrix}{V_{1} = \frac{\vartheta\; D^{2}H}{4}} & (1)\end{matrix}$where D is the diameter of the well and H is its height. Duringevaporation of the aqueous solvent, the glucose solution did not dewetfrom the freshly plasma-oxidized, hydrophilic surface. Consequently aconformal coating formed lining the inside of the microwells. If thecontour of the glucose layer is regarded as elliptical in shape, thevolume of empty space in the well that is not occupied by solid glucoseis:

$\begin{matrix}{V_{2} = \frac{\vartheta\; D^{2}h}{6}} & (2)\end{matrix}$where h is the height of microwell above solid glucose. If thevolumetric concentration of glucose is c, then

$\begin{matrix}{{cV}_{1} = {V_{1} - V_{2}}} & (3) \\{Thus} & \; \\{\frac{h}{H} = \frac{3( {1 - c} )}{2}} & (4)\end{matrix}$

When c>0.33, then h<H, which means the well is fully covered withglucose. When c 0.33, then h>H, which means the well is only partiallycovered with glucose (FIG. 5 ). The images are consistent withtheoretical prediction that the concentration of the glucose solutionmay be modified in order to generate full coverage of the interiorsurface of the microwells. Based on equation (4), the relative coverage(h/H) is dependent only on the concentration of glucose c, not on thedimension of microcavities (D and H). Therefore, in one embodiment, themicrocavities of a microfabricated device can be fully primed by using aglucose solution with c>33%, even for a surface possessing microcavitieswith a wide range of sizes (10 μm-3 mm) and depths.

As described above, glucose and sorbitol were used. as a sacrificialresidue for guiding wetting of water (or aqueous solution) inmicrocavities, corners and dead ends. After wetting, it is desirable tocompletely remove glucose and sorbitol from the chips without anyresidue left on the surface. Attenuated total reflection Fouriertransform infrared (ATR-FTIR) spectroscopy is a surface-sensitivediagnostic technique which can detect a trance amount of molecules onthe top surface (0.5-5 μm) of a sample. A flat PDMS sample was oxidizedwith plasma for 2 min and then primed with a thin layer of glucose orsorbitol in the same way as was used to prime microwell arrays. The PDMSsample was dried in air and stored at room temperature for 7 days. Thecoated PDMS sample was soaked in DI water for 5 min to dissolve glucoseor sorbitol, rinsed with DI water ×5 times, and dried in air. The. PDMSsample was then characterized by an ATR-FTIR spectrometer (Nicolet iS10,Thermo Scientific) with a Zinc Selenide (ZnSe) crystal to detect thepresence of glucose or sorbitol residue on the PDMS surfaces (FIG. 6 ).A native PDMS sample was used as a negative control, and glucose andsorbitol were used as positive controls. The characteristic peaks ofglucose (1338, 1223, 1199 and 1147 cm⁻¹) and sorbitol (935 cm⁻¹) wereabsent on coated PDMS samples (after water rinse). The spectra of coatedPDMS samples (after water rinse) were exactly same as the native PDMSsample. These results demonstrate that glucose and sorbitol residues onPDMS surface were not detectable. PDMS is well known to absorb smallhydrophobic molecules such as rhodamine B and Nile red (M. Toepke and D.Beebe, Lab Chip, 2006, 6, 1484-1486 (2006)), Glucose and sorbitol arehighly hydrophilic molecules (glucose has four hydroxyl groups andsorbitol has five hydroxyl groups) so that they are unlikely to beabsorbed by PDMS. Glucose and sorbitol are neutral molecules without anycharged and reactive functional groups, so that they are unlikelyadsorbed on the PDMS surface via electrostatic or covalent interactions.Because no residue is left in chips, glucose and sorbitol are idealsacrificial residues for guiding wetting of water (or aqueous solution)in microcavities, corners and dead ends.

In the above examples, glucose has been shown to be effective to coverthe PDMS microcavities and guide their wetting. Among ten differentpolymers of interest for microfabrication, PDMS. has the most rapidhydrophobic recovery (V. Jokinen et al., Biomicrofluidics, 2012, 6,016501 (2012)). Therefore, dissolution guided wetting using a glucosesacrificial residue on microwells made with other materials such aspolystyrene should be as effective as, if not easier than, in PDMS. Todemonstrate the glucose-guided wetting is applicable to microwells madefrom other materials, a microwell array was fabricated from polystyreneby our recently reported soft lithography micromolding technique (Y.Wang et al., Lab Chip, 2011, 11 3089-3097 (2011)), The wells had adiameter of 50 μm and a height of 55 μm. The polystyrene microwell arraywas oxidized with air plasma for 2 min, primed with a 37 vol % glucosesolution in the same way as with microwell arrays formed from PDMS.After priming, the micro-wells were covered with glucose (FIG. 7 ).After storage at room temperature for 7 days, water was added to thepolystyrene microwell array to test the rewetting guided by glucose.Water quickly (˜20-30 s) and completely dissolved the glucose and no airbubbles were trapped in the microwells (FIG. 7 ). This resultdemonstrates that dissolution guided wetting using glucose as thesacrificial residue is applicable to microfabricated devices made frommaterials other than PDMS.

Since glucose is an energy source for microbial metabolism, end usersmay be concerned about bacterial or fungal contamination during storage,especially in a humid and non-sterile environment. This issue can beaddressed by sterilization after the coating is applied using gamma-rayirradiation or ethylene oxide. Sugars that are poor energy sources suchas sorbitol, xylitol or mannitol can also be used to replace glucose (K.Isotupa et al., Am. J. Orthod Dentofac. Orthop., 107, 497-504 (1995)).Dissolution guided wetting using sorbitol as the sacrificial residue inPDMS microwells has been successfully achieved. The wells have adiameter of 50 μm and a height of 55 μm. The PDMS microwell array wasoxidized with air plasma for 2 min, primed with a 40 vol % sorbitolsolution and dried in air. After priming, the microwells were coveredwith sorbitol (FIG. S4 ). After storage at room temperature for 7 days,water was added to the PDMS microwell array to test the rewetting guidedby sorbitol. Water quickly (˜20-30 s) and completely dissolved thesorbitol and no air bubbles were trapped in the microwells (FIG. 7 ).This result demonstrates the sorbitol functions as effectively asglucose in guiding rewetting in microwells. An additional advantage ofsorbitol over glucose is its high solubility in water. The solubility ofsorbitol in water is 220 g/100 mL water (equivalent to 59.6 vol %, whichis much higher than that of glucose (91 g/100 mL water, equivalent to37.1 vol %). Sorbitol's higher solubility is useful in filling deepmicrocavities or to guide wetting in dead ends in microfluidic devices.

As described above, the dissolution guided wetting in the corners anddead ends of microfluidic channels was demonstrated using microfluidicchips built by molding PDMS channels from a master mold and then bondingit with glass slides through plasma oxidation (FIG. 1 a ). Immediatelyafter plasma treatment and bonding, 5 μL of a monosaccharide solution(in some experiments, the monosaccharide was mixed with 200 μg/mL TRITCdextran) was added to one reservoir for the microfluidic channel (FIG. 1b ). Due to the hydrophilic surface of the freshly plasma bonded PDMS,the solution spontaneously wet the entire channel within a few minutes,even in corners and dead ends without trapped air bubbles. A hosesupplied with nitrogen was placed at the opposite reservoir and used topurge the main microfluidic channel (FIG. 1 c ). The solution flushedout at the reservoir was removed by aspiration. A drop of water wasadded to the reservoir of the main channel during purging to dilute themonosaccharide solution as it exited the channel. The fluid in thereservoir was quickly aspirated after purging. After purging, residualsolution remained trapped in the dead ends (FIG. 1 d ). The chips werethen stored at room temperature and the residual monosaccharide solutionin the device was allowed to gradually dry by evaporation (FIG. 1 e ).In these experiments, a 30 vol. % glucose solution was used to fill thecorners in a microfluidic channel and a 50 vol % sorbitol was used tofill the dead ends in a microfluidic channel.

In another embodiment, a sacrificial residue is applied in contact withgas-entrapping features on native, hydrophobic structured surfaces of amicrofabricated device. The shape of the sacrificial residue depositeddepends on the interfacial property of the structured surface and thesolution comprising the sacrificial residue material (FIG. 10 -A). Inmicrocavities, a parabolic shaped residue is formed when the solutioncan wet the solid with small contact angle 74. On the other hand, aflat, column shape is formed when the solution cannot wet the solid witha large contact angle θ (in other words, the solution dewets on thestructured surface). In this embodiment, PDMS is used with a 25 wt %glucose aqueous solution as the examples for the substrate of amicrofabricated device having a structured surface and glucose insolution as the sacrificial residue material, respectively.

The native PDMS surface is hydrophobic which tends to trap air bubblesin microcavities and other gas-entrapping features. The PDMS surface canbe oxidized with plasma treatment to change its surface to behydrophilic. A 25 wt % glucose solution is immediately added to thehydrophilic PDMS surface. Upon drying in air, glucose forms a parabolicshape in microcavities (FIG. 10 -B), which can effectively guide wettingwhen water is added to the surface. The parabolic shape is caused by thehydrophilic PDMS surface, on which the glucose aqueous solution wets andforms a small contact angle (θ<90°).

When PDMS is not oxidized, glucose forms a flat, column shape in PDMSmicrocavities (FIG. 10 -C). This is caused by the hydrophobic PDMSsurface, on which the glucose aqueous solution dewets and forms a largecontact angle (θ>90°). This flat, column shape is not effective inguiding wetting in microcavities.

By using an organic solvent or an organic/aqueous mixture as thesolvent, a parabolic shape of sacrificial residue can be applied to anative, hydrophobic PDMS surface with microcavities. Examples of organicsolvents that are compatible with PDMS are ethanol, isopropanol,dimethylformamide, gamma-butyrolactone (GBL), gamma-valerolactone, etc.Organic solvents have much lower surface tension than water (72.8dynes/cm), for example, ethanol (22.4 dynes/cm), isopropanol (23.0dynes/cm). As a result, these organic solvents can wet native PDMS (19.8dynes/cm) and form a small contact angle. A suitable material for asacrificial residue can be dissolved in the organic solvent or anorganic/aqueous mixture, and then applied to the native PDMS surfacewith microcavities. Examples of solvent-sacrificial residue materialpairs are: ethanol/water mixture (50/5 wt/wt)-glucoseisopropanol-polyvinyl alcohol, GBL-polyethylene glycol.

In another embodiment, a sacrificial residue can be directly depositedon structured surfaces having gas-entrapping features by utilizing theliquid-solid phase transition of the material used to form thesacrificial residue. For example, sorbitol (Acros Organics #132731000)has a melting point of 95° to 99° C. Sorbitol was heated at 100° C. tobecome a low-viscous liquid, and then it was applied to surfaces onwhich microcavities were present. Air bubbles were removed by applyingvacuum or pressure. Excess sorbitol liquid was removed by aspiration.The microcavities were filled with liquid sorbitol which solidified asthe device cooled to room temperature. Upon subsequent wetting, thesorbitol residue effectively guided wetting in microcavities bydissolution without air bubble entrapment.

In a further embodiment, a microraft array such as is described inAllbritton et al., Array of Micromolded Structures for Sorting AdherentCells PCT Application No. PCT/US2011/025018 filed Feb. 16, 2011, hereinincorporated by reference, is composed of a large number of micron-scaleelements made from rigid plastics such as polystyrene, termed rafts,positioned at the bottom of microwells made from polydimethylsiloxane(PDMS). The scheme for the microraft array is shown in FIG. 11 -A-i.Within the array, the rafts serve as releasable culture sites forindividual cells or colonies. Cells are plated on the array in the samemanner as a Petri dish, and the cells remain positioned on specificrafts while in culture so that single cells can expand into clonalcolonies. To isolate target cells, a needle is inserted through the PDMSto dislodge a raft and its attached cell(s). The cell(s) is thencollected for expansion, or downstream analysis.

Air bubbles are presented when water (or aqueous buffer, medium) isadded on the array. The trapping of air bubbles is caused by thehydrophobic surface of PDMS (FIG. 11 -A.-ii). The array can be treatedwith plasma to change the PDMS surface to be temporarily hydrophilic,but the PDMS surface can quickly recover its native hydrophobic naturein a few days. For example, two weeks after plasma treatment, airbubbles are presented on the array when water is added (FIG. 11 -A-iii).The air bubbles need to be removed since they prevent cells fromsettling into the wells and attaching to the rafts.

To prevent formation of air bubbles, glucose was used as a sacrificialmaterial and was deposited on the microraft array by applying a 40 wt %glucose solution on the plasma-oxidized microraft array followed byaspiration to remove excess solution and drying in air (FIG. 2 -B-i).The glucose coated raft, array can be stored under ambient conditionsfor at least weeks to months. Even after sufficient time (2 weeks afterplasma treatment and deposition of glucose residue) for the PDMS torecover its hydrophobic surface, the dissolution of glucose effectivelyguided wetting in wells when water was added (FIG. 11 -B-ii) as shown bycomplete absence of air bubbles on the array after water addition (FIG.11 -B-iii).

Glucose is nontoxic and serves as an energy source for cells. Glucose isone of the main components of cell culture medium, for example,Dulbecco's Modified. Eagle's Medium (DMEM) has a. glucose concentrationof 4.5 grams/liter. Glucose is perhaps one of the safest sacrificialresidues to guide wetting in microfabricated devices used forapplications involving biological cells. Glucose does not stay on thePDMS surface after rinsing with water as verified by attenuated totalreflectance (ATR) FTIR spectroscopy. To demonstrate the lack of adetrimental effect of a glucose sacrificial residue on cell culture, amicroraft array prepared with a glucose sacrificial residue as describedabove was rinsed with phosphate buffered saline (PBS) three times, thenplated with 111299 cells, a human non-small cell lung carcinoma cellline. The proliferation and morphology of cells was normal and identicalto those of cells cultured on a rnicroraft array without glucosesacrificial residue. This result demonstrated that the glucose residuehad no negative effect on the growth and health of the cultured cells.

A simple method has been described herein that prevents or eliminatesthe formation of gas bubbles in gas-entrapping features ofmicrofabricated devices, thus solving a common problem encountered whenfluids are added to such devices for a variety of lab-on-a-chipapplications. The method involves priming the structured surfaces of themicrofabricated device through the application of a sacrificial residuethat achieves the dissolution guided wetting of the gas-entrappingfeatures upon introduction of a suitable fluid for the intended use ofthe device. Microfabricated devices thus primed can be kept in drystorage for a prolonged period without loss of efficacy of thedissolution guided wetting. Gas bubble formation was prevented by simplyadding a suitable solvent or solution to the device to dissolve thesacrificial residue. Simple, robust, and easily implemented methods suchas this are needed to increase the rate of adoption of microwell arraysand microfluidic technologies in everyday laboratory practice as well asin the field and in clinical applications. The current techniquerepresents an example of combining robust simplicity with functionality.If a sacrificial residue is selected that can act as an energy sourcefor microbial metabolism, concerns about bacterial or fungalcontamination during storage can be addressed by sterilization of theprimed microfabricated device using gamma-ray irradiation or ethyleneoxide. Sugars that are poor energy sources such as sorbitol, xylitol. ormannitol can also be used to replace. glucose.³³ Dissolution guidedwetting can also be achieved with microfabricated devices havinghydrophobic structured surfaces by selecting hydrophobic solvents andsuitable solutes as the sacrificial residue. The method is applicable toa variety of polymer-based, lab-on-a-chip products with gas-entrappingfeatures including microwell arrays and enclosed microfluidic systemswhere surface wetting is particularly challenging.

The foregoing description and embodiments is illustrative of the presentinvention, and is not to be construed as limiting thereof. The inventionis defined by the following claims, with equivalents of the claims to beincluded therein.

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The foregoing: is illustrative of the present invention, and is not tobe construed as limiting thereof. The invention is defined by thefollowing claims, with equivalents of the claims to be included therein.

That which is claimed is:
 1. A microfabricated device comprising: asubstrate material fabricated to comprise a structured surface formedtherein, at least one gas-entrapping feature on the structured surfacethat entraps gas bubbles upon wetting said structured surface with anaqueous or non-aqueous solvent or solution, wherein the substratematerial comprises an array of microwells formed therein; and asacrificial residue in contact with the at least one gas-entrappingfeature, wherein the sacrificial residue is soluble in an aqueous ornon-aqueous solvent or solution, wherein the sacrificial residue isconfigured to cause dissolution guided wetting upon contact with theaqueous or non-aqueous solvent or solution to thereby fill the at leastone gas-entrapping feature without entrapping gas bubbles.
 2. The deviceof claim 1, wherein said gas-entrapping feature comprises a well,corner, microcavity, dead end, post, trap, hole, passage, channel, orcombination thereof.
 3. The device of claim 1, wherein said device iscomprised of an organic polymer.
 4. The device of claim 1, wherein saidsubstrate is comprised of a polymer selected from the group consistingof as polymethylmethacrylate (PMMA), polycarbonate,polytetrafluoroethylene (PTFE), polyvinylchloride (PVC),polydimethylsiloxane (PDMS), polysulfone, polystyrene,polymethylpentene, polypropylene, polyethylene, polyvinyl idinefluoride, acrylonitrile-butadiene-styrene copolymer (ABS), polymerizedphotoresists, and combinations thereof.
 5. The device of claim 1,wherein the sacrificial residue is dissolvable in the aqueous solvent orsolution and is comprised of a salt, carbohydrate, hydrophilic polymer,dextran, polyethylene glycol, alginate, agarose, chitosan, glucose,sucrose, non-metabolizable carbohydrate, or sorbitol.
 6. The device ofclaim 1, wherein the sacrificial residue is dissolvable in thenon-aqueous solvent or solution and is comprised of a hydrophobicpolymer, low molecular weight organic solid, non-volatile liquid,polyethylene, polypropylene, polyvinyl chloride, polystyrene, nylon,polyester, acrylic, polyurethane, polycarbonate, polylactic acid,poly(lactic-co-glycolic acid), poly(methyl methacrylate), paraffin wax,naphthalene, anthracene, aspirin (acetylsalicylic acid), 2-naphthol,fat, synthetic oil, mineral oil, vegetable oil, olive oil, lipid,silicone oil, or liquid epoxy resin.
 7. A method of priming amicrofabricated device for dissolution guided wetting of gas-entrappingfeatures, comprising: (a) providing a microfabricated device comprisinga substrate material fabricated to comprise a structured surface formedtherein, at least one gas-entrapping feature on the structured surfacethat entraps gas bubbles upon wetting the structured surface with anaqueous or non-aqueous solvent or solution, wherein the substratematerial comprises an array of microwells formed therein; (b) applying asacrificial residue to the at least one gas-entrapping feature on thestructured surface, wherein the sacrificial residue is soluble in anaqueous or non-aqueous solvent or solution, wherein the sacrificialresidue becomes dissolved in the aqueous or non-aqueous solvent orsolution upon wetting the structured surface thereby inhibiting theentrapment of gas bubbles therein through dissolution guided wetting. 8.The method of claim 7, wherein the sacrificial residue comprises asolute dissolved or dispersed in a second solvent or solution and theapplying comprises contacting the solute dissolved or dispersed in thesecond solvent or solution with the at least one gas-entrapping feature,and allowing the second solvent or solution to dry thereon therebydepositing the solute in contact with the gas-entrapping feature.
 9. Themethod of claim 7, wherein the sacrificial residue is applied to the atleast one gas-entrapping feature as a fine particulate dust.
 10. Themethod of claim 7, wherein the device is comprised of an organicpolymer.
 11. The method of claim 7, wherein the substrate is comprisedof a polymer selected from the group consisting of aspolymethylmethacrylate (PMMA), polycarbonate, polytetrafluoroethylene(PTFE), polyvinylchloride (PVC), polydimethylsiloxane (PDMS),polysulfone, polystyrene, polymethylpentene, polypropylene,polyethylene, polyvinylidine fluoride, acrylonitrile-butadiene-styrenecopolymer (ABS), polymerized photoresists, and combinations thereof. 12.The method of claim 7, wherein the sacrificial residue is dissolvable inthe aqueous solvent or solution and is comprised of dextran,polyethylene glycol, alginate, agarose, chitosan, glucose, sucrose,non-metabolizable carbohydrate, or sorbitol.
 13. The method of claim 7,further comprising treating the microfabricated device with plasmaoxidation prior to applying the sacrificial residue to the at least onegas-entrapping feature on the structured surface.
 14. The method ofclaim 7, wherein the sacrificial residue is dissolvable in thenon-aqueous solvent or solution and is comprised of a hydrophobicpolymer, low molecular weight organic solid, non-volatile liquid,polyethylene, polypropylene, polyvinyl chloride, polystyrene, nylon,polyester, acrylic, polyurethane, polycarbonate, polylactic acid,poly(lactic-co-glycolic acid), poly(methyl methacrylate), paraffin wax,naphthalene, anthracene, aspirin (acetylsalicylic acid), 2-naphthol,fat, synthetic oil, mineral oil, vegetable oil, olive oil, lipid,silicone oil, or liquid epoxy resin.
 15. The method of claim 7, whereinthe microwells further comprise a releasable element positioned at thebottom of each of the microwells.
 16. A method of wetting amicrofabricated device while inhibiting the entrapment of gas bubblestherein, comprising: (a) providing a microfabricated device comprising asubstrate material fabricated to comprise a structured surface formedtherein, wherein the substrate material comprises an array of microwellsformed therein, at least one gas-entrapping feature on the structuredsurface that entraps gas bubbles upon wetting the structured surfacewith an aqueous or non-aqueous solvent or solution, and a sacrificialresidue in contact with the at least one gas-entrapping feature, whereinthe sacrificial residue is soluble in an aqueous or non-aqueous solventor solution; and (b) treating the microfabricated device with theaqueous or non-aqueous solvent or solution sufficient to dissolve andremove the sacrificial residue from the at least one gas-entrappingfeature while concurrently wetting the at least one gas-entrappingfeature with the solvent or solution thereby inhibiting the entrapmentof gas bubbles therein through dissolution guided wetting.
 17. Themethod of claim 16, wherein the aqueous solvent or solution comprises agrowth media, an assay or reagent media, or a reaction media.
 18. Themethod of claim 16, wherein the gas-entrapping feature comprises a well,corner, microcavity, dead end, post, trap, hole, passage, channel, orcombination thereof.
 19. The method of claim 16, wherein the microwellsfurther comprise a releasable element positioned at the bottom of eachof the microwells.
 20. The method of claim 16, wherein the device iscomprised of an organic polymer.
 21. The method of claim 16, wherein thesubstrate is comprised of a polymer selected from the group consistingof as polymethylmethacrylate (PMMA), polycarbonate,polytetrafluoroethylene (PTFE), polyvinylchloride (PVC),polydimethylsiloxane (PDMS), polysulfone, polystyrene,polymethylpentene, polypropylene, polyethylene, polyvinylidine fluoride,acrylonitrile-butadiene-styrene copolymer (ABS), polymerizedphotoresists, and combinations thereof.
 22. The method of claim 16,wherein the sacrificial residue is dissolvable in the aqueous solvent orsolution and is comprised of a salt, carbohydrate, hydrophilic polymer,dextran, polyethylene glycol, alginate, agarose, chitosan, glucose,sucrose, non-metabolizable carbohydrate, or sorbitol.
 23. The method ofclaim 16, wherein the sacrificial residue is dissolvable in thenon-aqueous solvent or solution and is comprised of a hydrophobicpolymer, low molecular weight organic solid, non-volatile liquid,polyethylene, polypropylene, polyvinyl chloride, polystyrene, nylon,polyester, acrylic, polyurethane, polycarbonate, polylactic acid,poly(lactic-co-glycolic acid), poly(methyl methacrylate), paraffin wax,naphthalene, anthracene, aspirin (acetylsalicylic acid), 2-naphthol,fat, synthetic oil, mineral oil, vegetable oil, olive oil, lipid,silicone oil, or liquid epoxy resin.